Receptor binding assays have been commonly employed for studying receptor ligand interactions in e.g. neurochemistry, neurobiology. psychopharmacology and related fields. Receptor assays are also used as an analytical method to measure drug levels in biological matrices. The principle of these methods is based on the competition of a labeled ligand and the analyte for binding to a certain receptor. Up till now, receptor assays have mostly been performed with radioactive ligands. Due to the low density of receptor binding sites in most tissue e.g. 10 to 100 pmol ligand bound per gram of tissue the ligand used must have high affinity and selectivity for the binding sites as well as high specific radioactivity in order to determine low levels of ligand. Radioactive ligands are commercially available that can be selectively measured at very low levels due to their high specific activity. Besides, since most of the radioisotopes are incorporated in the molecule, this type of labeling has no influence on the binding affinity of the ligand towards the receptor. Since the use of radioactivity has several well documented disadvantages such as limited shelf life, problematic handling and care required in the disposal thereof, non-radioactive ligands, such as fluorescent-labeled ligands, have been synthesized for different receptor types. To date however the results have not been such that reliable quantitative data for trace amounts of analytes can be obtained in a simple and satisfactory manner that they can offer an alternative for those types of analysis carried out with radioactive receptor assays (RRAs).
Examples of a non radioactive receptor assay are provided in general for a large number of receptors in WO93/03382 of Tyler McCabe. They describe how numerous attempts to characterize receptors using fluorescent ligands were carried out. The references cited on page 3 of the PCT application are hereby also incorporated by reference. The receptors mentioned are xcex1-adrenergic xcex2-adrenergic, opioid, adenosine, glucagon, steroid and dopamine receptors. They indicate there were problems with quantification and visualisation by direct fluorescence measurement due to autofluorescence and lack of specificity. They provide a group of fluorescent ligands suitable for determining ligand-receptor interactions intracellularly and extracellularly and to determine the specificity and affinity of uncharacterised compounds. They analyse intracellular versus extracellular events by selecting a fluorescent probe which emits different fluorescent intensities depending on the pH of the environment. They carry out their tests on 100% tissue. The receptors mentioned were opioid, potassium channel, glibenclamide and glycine. The assays actually illustrated are a fluorescein labelled ligand for the opioid receptor, nitrobenz-2-oxa-1,3-diazol-4-yl(=NBD) labelled ligands for the potassium channel and glibenclamide and glycine receptors. They indicate the fluorescent value is corrected for by subtracting the value of auto fluorescence. This means the assay they employ can never be sufficiently sensitive to obtain reliable quantification or detection of trace amounts of analytes.
One of the important receptors for pharmacologists and doctors is the benzodiazepine receptor. Benzodiazepines are extremely widely used drugs and have been primarily used for the treatment of anxiety and insomnia. It is accepted the pharmacological effects are medicated through specific receptors in the central nervous system. This receptor has been found to be extremely difficult to tackle for determining low concentrations of benzodiazepine receptor binding analytes. In vitro receptor radioligand interactions have been used to investigate the mechanism of the pharmacological effects and also to investigate new benzodiazepine drugs.
An alternative method has been the use of non radioactive immunoligand assays. These solve the problems associated with the use of radioactivity. The disadvantages thereof are however numerous. In immunoassays the binding molecule is an antibody that has been generated against the ligand to be determined. In order to generate antibodies the ligand must first be linked to a large carrier for example BSA. The position of the linkage of the BSA to the ligand restricts the selectivity of the antibody. The antibody is not selective for the linkage position. The sensitivity of immunoassays is not correlated to the pharmacological effectivity of the ligand. The affinity between ligand and antibody is based merely on chemical structure not an the chemical structure that determines the pharmacological effectivity. A ligand can have a high affinity for the antibody whet such Ligand is hardly pharmacologically effective and vice versa. For example the Merck label for the fluorescent polarisation immunoassay for benzodiazepines (Vitalab Eclair) hardly has any affinity for the benzodiazepine receptor (Ki=200 nM). With regard to metabolites nothing can be stated with regard to their pharmacological activity. In addition when multiple ligands need to be determined multiple antibodies are required for each separate ligand. In the receptor ligand assays a single ligand can suffice to assay for multiple analytes. The immunoligand technology is for example illustrated in EP-A-0.264.797 of Abbott.
Specifically for the benzodiazepine receptor for example fluorescent-labeled ligands have also been used as non radioactive labeled ligand for the characterization of the benzodiazepine receptor [g-i]. Such characterisation experiments however are not subject to the degree of sensitivity required for analyte detection and quantification at trace limits. The content of the cited articles will however be presented furtheron in order to create a more complete overview of the technology and the specific problems.
Further to the above more recent publications for the benzodiazepine receptor address use of fluorescent-labeled ligands as labeled ligand for a benzodiazepine receptor assay [b (1991),j (1993). c(1995)] as opposed to simple characterisation.
In the fluorescent benzodiazepine receptor assays developed by Takeuchi et al. [b,c], the free fractions of label were quantified after collection of these fractions by centrifugation. Since membrane-bound receptors exhibit background fluorescence [c] the problem of autofluorescent interference was expected to be reduced in these assays. However this was not the case. Additional measures were required and the sensitivity of these assays left a lot to be desired,
Takeuchi and Rechnitz in [b] already in 1991 described quantifying the free fraction of the ligand. They used a HPLC-system in conjunction with AMCA-Ro7-1986 (AMCA-didesethylflurazepam) as ligand in their fluorescence receptor assay. The use of HPLC was required as a pretreatment to eliminate possible interference in the matrix. The supernatant after the bound/free separation by centrifugation was injected directly onto the HPLC column without further cleanup such as filtration. To obtain enough difference in the fluorescence signal between the maximal binding and the non-specific binding, they also had to use a high amount of receptor material, 50 mg/ml. Such a test is not practical for large scale commercial use due to the prohibitive cost of using such high amounts of receptor material. In addition the use of such high amounts of animal tissue is undesirable also from an ethical point of view.
Takeuchi et al. [j and c] later addressed the problem of autofluorescence in an alternative and preferred manner as disclosed in their articles of 1993 and 1995. Specifically they stated in the latter article xe2x80x9cBecause the commercially available benzodiazepine receptor preparations are only partially purified their supernatants exhibit strong background fluorescence and may interfere with the measurement of fluorophore labeled ligandsxe2x80x9d. To solve this problem in the cited article they disclose developing a time-resolved fluorometric assay for benzodiazepines. They specifically state xe2x80x9cradioligand receptor assays are frequently performed in laboratories as no feasible nonisotopic assays using drug receptors have been reportedxe2x80x9d. They also provide the considerations for the development of nonisotopic receptor assays xe2x80x9cthe label may not significantly reduce the ligand affinity to the receptor land at the same time a highly sensitive measurement method for the labeled ligand must be available so sensitivity comparable to radioisotopic methods can be achieved.xe2x80x9d Their new process involved selection of a specific label with special fluorescent capabilities. They chose to use a europium chelate as label, since their supernatant exhibited strong background fluorescence which interfered with the measurement of the fluorophore-labeled ligands. The europium chelate provides a different type of fluorescence than the autofluorescence of the membrane material. The europium chelate provides a long lifetime fluorescence after excitation with pulsed light. This enables performance of time resolved fluorometry without interference front short term lifetime fluorescence of common fluorophores such as those present in the receptor matrix. They subsequently separated the bound and free fractions of their label, Eu-1021-S, by centrifugation and quantified the free fractions by the measurement of time-resolved fluorescence in the supernatant. The subject invention provides a suitable alternative and furthermore enables use of fluorescent labels that can also be determined at wavelengths in the area of the background fluorescence. The subject invention does not require time resolved fluorescence. The subject invention reaches higher sensitivity. To obtain enough difference in the fluorescence signal between the maximal binding and the non-specific binding, Takeuchi et al. also had to use a high amount of receptor material, 50 mg/ml. Such a test is no practical for large scale commercial use due to the prohibitive cost of using such high amounts of receptor material. In addition the use of such high amounts of animal tissue is undesirable also from an ethical point of view. As referred to above articles [g-i] dealt with characterisation of benzodiazepine receptors rather than quantification. Havunjian et al. [g] and McCabe et al. [h] do however describe how they quantified the bound fractions of their labelled ligands. However as disclosed above since membrane-bound receptors exhibit background fluorescence [c] and their measurements were executed in the presence of the receptor materials additional measures needed to be taken to overcome this problem. Specifically Havunjian used fluorescent-labeled benzodiazepine BD 623 (NBD-NHxe2x80x94(CH2)3-Ro15-3890, also known as NBD-desethylflumazenil) as a benzodiazepine ligand in assays monitoring fluorescence/dequenching. The bound fraction was determined. After determining the autofluorescence of the membrane preparation (autofluorescence) (region A, FIG. 2A of the cited article) BD 623 was added and fluorescence was monitored over time. Fluorescence was gradually quenched to a plateau (region C) when addition of excess flurazepam effected a dequenching of fluorescence that was monitored to equilibrium (region D). The use of SD 623 as a prototype for the development of other fluorescent ligands to study ligand receptor interactions was postulated. In practice the assays were found to be insufficiently sensitive to provide an assay capable of detecting low amounts of analyte. They required high amounts of ligand, in the example provided the amount of ligand works out at approximately 10*Kd. They also required high amounts of receptor material. Havunjian discloses xe2x80x9cThe use of receptor densities 20-80 fold higher in fluorescence compared to radioreceptor assays . . . xe2x80x9d. In this method, it is remarkable that they can detect the amount of quenching of NBD-NHxe2x80x94(CH2)3-Ro15-3890, specially regarding the low quantum yield of the fluorophore NBD, which is 0.02 in Tris-citrate buffer (pH 7.4; 50 mM) [g]. They did not present a feasible alternative to radio receptor ligand assays.
The Tyler McCabe article [h] (of a later date than the previously cited PCT application of this author) addresses the problems associated with radioassays and postulates the application of fluorescence as an alternative. Two benzodiazepine ligands labeled with fluorophores are presented fluorescein-NHxe2x80x94(CH2)3-Ro15-3890 (BD 621) and (BD 607) the direct coupling product of Ro-7-1986 with carboxyfluorescein-N-hydroxysuccinimide ester in DMF. BD 621 is a fluorescein desethylflumazonil derivative and BD 607 is a fluorescein didesethylflurazepam derivative. McCabe et al. measured the bound fractions of their fluorescent-labeled benzodiazepine. After separation of the bound and unbound fractions by centrifugation, they resuspended the pellet in buffer and measured the fluorescence intensity of the suspension. The fluorescently labelled ligand was still bound to the receptor during the measurement. No indication of detection limit is provided. High amounts of ligand and high amounts of receptor for a sensitive assay following the teaching of this document would be envisaged. It is remarkable their fluorescent-labeled benzodiazepine could in fact even be detected in the presence of the receptor material. However, fluorophore fluorescein has relatively high excitation and emission wavelengths (xcexex=499 nm and xcexem=521 nm) in comparison to other fluorophores, such as a coumarin derivate. The autofluorescence of the receptor material is less at higher wavelengths, which could explain the matter. Nevertheless the interference will still be enormous. They themselves even found that the fluorescence intensity of fluorescein-NHxe2x80x94(CH2)3-Ro15-3890 was stronger when measured in tissue suspension than when measured in buffer only and that this increased intensity was not due to the background fluorescence of the tissue suspension. The Kd values of the fluorophores illustrated are 63 and 74 which render them incapable of sufficient sensitivity for application in detecting and quantifying trace analytes. In practice tho assays were insufficiently sensitive to provide an assay capable of detecting low amounts of analyte and required high amounts or receptor material.
It is an objective of the subject invention to provide a highly sensitive receptor-ligand assay that does not require dealing with radioactivity but provides a test with at least the sensitivity and specificity of a radioactive receptor assay. Such an assay must be simple to execute and economically feasible enabling routine laboratory application.
The subject invention provides a highly sensitive receptor-ligand assay that does not require dealing with radioactivity but provides a test with at least the sensitivity and specificity of a radioactive receptor assay. Such an assay is simple to execute and economically feasible enabling routine laboratory application. Also a group of ligands specifically directed at benzodiazepine receptors suitable for use in an essay according to the invention is provided.